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involved in the transcription of miRNAs, interfere with the miRNA
maturation process, or inhibit incorporation of the mature miRNA
into RISC.14 Thus, the discovery of small-molecule modulators of
miRNA function has gained significant momentum in recent
years.15–24
assays. In recent literature, a concern for the presence of firefly
luciferase inhibitors in large compound libraries and the conse-
quential appearance of false positives has been reported.25 The
firefly luciferase enzyme is sensitive to ligand-based stabilization
by small molecule inhibitors; therefore in a cell-based assay, treat-
ment with an inhibitor can lead to an increase in luciferase signal
after cell lysis and assay readout. Conversely, biochemical evalua-
tion of the same inhibitors causes a decrease in luciferase signal.25
In particular, compound 28 has been previously identified as a fire-
fly luciferase inhibitor in a biochemical qHTS (PubChem AID 411).
In order to confirm that the miR-21 inhibitors are not in fact
inhibiting luciferase, an in vitro luciferase assay was employed.
Treatment of firefly luciferase enzyme with 3, 9, 23, or 28 at
10 lM showed no effect on luciferase activity validating that these
compounds do not inhibit luciferase enzyme (SI Fig. 1).
To investigate each inhibitor’s selectivity for miR-21, com-
pounds 3, 9, 24, and 28 were tested at 10 lM in our previously
described Huh7-psiCHECK-miR122 reporter cell line, which
expresses Renilla luciferase under the control of miR-122 activity.16
None of the four inhibitors displayed any effect on luciferase
expression in this secondary assay, while showing a greater than
100% increase in luciferase expression in the HeLa-miR21-Luc cell
line (Fig. 2A). This provides evidence that these compounds display
(some level of) specificity to miR-21 and are not general inhibitors
of the miRNA pathway. Furthermore, satisfactory dose response
curves were generated for each compound in the HeLa-miR21-
Previously, we identified a selective small-molecule inhibitor of
miR-21 from a pilot screen in HeLa cells using a luciferase-based
reporter assay that places luciferase expression under the regula-
tion of miR-21 activity.15 In order to identify additional, struc-
turally diverse small molecule miR-21 inhibitors, an extensive
high-throughput screen of 333,519 compounds was performed
(pubchem AID 2289), which delivered 3282 hits (1% hit rate).
Subsequently, 124 of the top ranking small molecules identified
in the primary assay were submitted to secondary screens to eval-
uate their selectivity, as well as their specificity. The compounds
were tested in a cell-based assay expressing a reporter construct
targeting miR-30a (pubchem AID 2507). Compounds active in both
the miR-21 and miR-30a assays were disregarded as they were not
considered selective for miR-21 inhibition, but rather may be gen-
eral miRNA modulators or non-specifically target firefly luciferase.
Additionally, the initial hits were subjected to an in vitro firefly
luciferase assay using recombinant firefly luciferase enzyme (pub-
chem AID 493175). Compounds identified as firefly luciferase inhi-
bitors were disregarded as false positives (see detailed discussion
below). Ultimately, 58 small molecules were confirmed as inhibi-
tors of miR-21. After collecting dose–response data and qRT-PCR
results on the down-regulation of miR-21 levels, the aryl amide 1
was selected for follow-up SAR studies.
Luc assay revealing EC50 values of 10.8
0.86 M, respectively (Fig. 2B).
lM, 6.1 lM, 2.3 lM, and
l
Following re-synthesis and confirmation of 1, structure–activity
relationship studies were conducted through the synthesis and
testing of analogs (Fig. 1). Replacing the bulky piperidine moiety
with a smaller acetyl (2) or methoxy (3) group resulted in favorable
47% and 65% increases in activity, respectively. Further modifica-
tion of the meta position to a hydroxyl group (4) showed a more
modest 27% increase in activity over the parent compound 1, while
removal of the meta substituent altogether (5) completely abol-
ished activity. Interestingly, modification of the para or meta posi-
tions to hydroxyl groups (6, 7) or removal of both substitutions (8)
resulted in very little change to the activity, while the 1,3-disub-
stitued hydroxyl derivative (9) displayed a very promising 86%
increase in activity. Modification of 4 from a cyclohexene to a
cyclohexane (10) resulted in a 57% increase in activity relative to
1, while both its benzene derivative (11) or loss of the ring entirely
(12) resulted in complete loss of activity. Surprisingly, modification
of the p-methoxy to an ethyl ether (13), propyl ether (14), benzyl
ether (15), or amine (16) rescued activity. Retaining the p-methoxy
while extending the aliphatic linker by an additional carbon (17)
yielded a 58% increase in activity relative to the parent compound
1. Based on the identification of 17, several new analogs were syn-
thesized that modified the benzene ring and its aliphatic linker.
Extending or shortening the linker by an additional carbon (18,
19) resulted in complete loss in activity. Modification of the ben-
zene ring in 19 to a 2-pyridine (20) also resulted in a loss in activ-
ity. Introduction of a naphthalene (21) rescued partial activity, as
did removing an additional carbon from the linker (22).
Completely removing the aliphatic linker (23) resulted in a drastic
98% increase in activity over 1. Replacement of the benzene ring
with a naphthalene (24), a propylene (25), or the free amide (26)
only reduced the activity. Removal of the p-methoxy (27) from
23 also resulted in a loss in activity, but introduction of an addi-
tional p-methoxy on the benzene ring (28) resulted in a derivative
with a 152% increase in activity over the original hit compound 1.
Based on the results from the SAR investigation, four of the most
potent aryl amides 3, 9, 23, and 28 were further investigated.
While highly sensitive, the use of a luciferase-based assay in a
HTS requires validation of hit compounds through secondary
Inhibitors 3, 9, 23, and 28 were then analyzed by quantitative
RT-PCR to measure their direct effect on miR-21, miR-125b, miR-
17-5p, and miR-222 expression levels in HeLa cells. Cells were
incubated with 3, 9, 23, or 28 at 10 lM for 48 h, the total RNA
was isolated (miRNeasy kit, Qiagen), and the quantification was
performed with the corresponding TaqMan microRNA assays
(Life Technologies). While 3 showed only a small 17% reduction
in miR-21 expression, the inhibitors 9, 23, and 28 showed more
significant reductions of 42%, 43%, and 61%, respectively (Fig. 3A).
These values correlate well with their respective EC50 values deter-
mined in the HeLa-miR21-Luc assay. Additionally, all four inhibi-
tors showed little or no effect on miR-125b, miR-17-5p, or miR-
222 levels, providing additional support that these compounds
may be specific to miR-21.
To explore a possible mode of action for the inhibitors, RT-PCR
was used to specifically quantify the expression levels of primary-
miR-21 in HeLa cells. Following treatment with 3, 9, 23, and 28, lit-
tle to no effect on primary-miR-21 levels was observed, suggesting
that these inhibitors may act downstream of transcription of the
miR-21 gene (Fig. 3B). This is in contrast to previously discovered
small molecule inhibitors of miRNA function and may lead to fun-
damentally new chemical probes to interrogate the miRNA path-
way.15,16,20,21 To further confirm this, the endogenous miR-21
promoter was cloned upstream of a luciferase gene in the pGL4
construct (Promega), thereby placing the reporter under control
of the miR-21 promoter. Transfection of the pGL4-miR21P plasmid
into HeLa cells resulted in a >3000-fold increase in luciferase signal
compared to the parent pGL4-empty vector (Fig. 4A). Next, HeLa
cells transfected with the pGL4-miR21P vector were treated with
DMSO or inhibitors 3, 9, 23, or 28, followed by a luciferase assay
to determine reporter gene expression. As expected, treatment
with the inhibitors had no effect on the reporter gene and thereby
the miR-21 promoter (Fig. 4B), further supporting that these inhi-
bitors do not effect the transcription of miR-21, but may rather
inhibit the miR-21 maturation pathway. Although quite unlikely
due to the relatively small and planar core structures of the aryl-
amide miR-21 inhibitors, another possibility would be that these
compounds inhibit miR-21 by direct interaction with the miRNA.